The Response of Individual Living Cells of Chlamydomonas Spp. to High Light and Singlet Oxygen
Date
2018-07-13
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
ORCID
0000-0002-1913-3968
Type
Thesis
Degree Level
Doctoral
Abstract
Oxidation-reduction reactions are critical to life on earth as we know it, via photosynthesis and/or respiration. As life becomes more complex, these reactions become essential to gene expression, chemosignalling, and physiological stress responses. Imbalance in these reactions can result in disruption of these pathways, as well as damage to cellular components ranging from lipids to proteins to DNA. Photosynthetic organisms are especially prone to these conditions, given the nature of the reactions used to convert light energy to chemical energy. Exposure to high light (HL) results in the production of a number of dangerously reactive byproducts, chief amongst them singlet oxygen. We have a good understanding of how HL results in singlet oxygen production and a rudimentary knowledge of organisms’ genetic responses. However, the response of individual living cells at the biochemical level following HL stress remains unexplored, largely due to a lack of tools.
In this thesis, green algae of three species of Chlamydomonas (reinhardtii, DJX-J, and DJX-H) and four photosynthetic mutants of C. reinhardtii were exposed to high light, the singlet-oxygen producing photosensitizer rose bengal (RB), or a combination of HL and RB. Synchrotron-based Fourier Transform Infrared Spectromicroscopy (FTIR) was used to study the biochemical response of individual living cells to these exposures, as well as to characterize differences between cell lines. The FTIR system I developed was able to discriminate between different species of Chlamydomonas, between the C. reinhardtii mutants, and even between different cultures of the same species.
The synchrotron-based FTIR system allowed me to observe biochemical responses to stress exposures. These were primarily in peaks related to proteins (changes in secondary structure) and lipids (changes in diversity, membrane fluidity, lipid oxidation byproducts, degree of unsaturation, and changes in membrane function). Observed in vivo biochemical changes in living cells were dependent upon species/photosynthetic mutation and route of stress exposure. Measurements were able to differentiate between cells exposed to HL, RB, and the combination of both. Exposure to RB/HLRB was more likely to result in observable changes in membrane function.
Overall, this work clearly demonstrates the power of FTIR in studying living systems. Not only could I discriminate between very similar cells, but it provided the first assessment of the biochemical responses of individual living cells to high light, rose bengal, and the combination of these factors.
Description
Keywords
Chlamydomonas reinhardtii, singlet oxygen, high light, oxidative stress, Chlamydomonas, FTIR, Fourier-transform infrared spectromicroscopy, Fourier-transform infrared microspectroscopy, synchrotron FTIR, cell biology
Citation
Degree
Doctor of Philosophy (Ph.D.)
Department
Biology
Program
Biology